FIGS. 1a and 1b illustrate two types of light illuminations. FIG. 1a illustrates Bright Field illumination 10. Bright Field illumination is present when the aperture of the objective 12 (of the microscope) is smaller or equal to the aperture of the condenser (not shown). A main feature of Bright Field illumination 10 is that all light 14 should go into the objective.
FIG. 1b illustrates Dark Field illumination 16. Dark Field illumination 16 is present when the aperture of the objective 12 is smaller than aperture of the condenser. Direct light coming out of the condenser does not go into the objective 12 in Dark field illumination 16. However, misalignment of the condenser, for example, can introduce background light or noise into the system, which reduces contrast. Therefore, circular symmetry of the illumination system is required for Dark Field illumination 16.
Dark Field illumination 16 has some distinct advantages over Bright Field illumination 10. Dark Field illumination 16 has much higher contrast and better light economy than Bright Field illumination 10.
FIG. 1c illustrates the alignment issues involved with the Dark Field Microscope in FIG. 1b. The light source 18, condenser 20, and axicon lens 22 must be perfectly aligned such that the angle of the light path 14 intersects and illuminates the sample properly as will be explained in further detail herein. It should be noted that FIGS. 1b and 1c have been simplified from FIG. 1a in that both FIGS. 1a and 1b do not show the condenser, axicon lens, nor the light source 18, but the Bright Field and Dark Field Microscopes depicted in FIGS. 1a and 1b, respectively, can include such elements.
Various methods of illumination are employed in Dark Field Illumination 16. Two commonly used methods are Critical illumination and Kohler illumination. FIG. 2a illustrates the Critical illumination 24. The system includes a uniformly bright light source 18, a diaphragm 28, a condenser aperture 26, a condenser 20, a slide 30, a sample 32 coupled to the slide 30, and a microscope objective 12. In this method of illumination, the uniformly bright light source 18 is placed close behind the diaphragm and is imaged by the condenser 20 on to an object plane of the microscope objective 12. The size of the field stop aperture is adjusted so that its image by the condenser 20 just covers the field. The complex degree of coherence for any pair of points in the object lance of the objective 12 is the same as that due to an incoherent source of filling the condenser aperture 26; moreover it is independent of the aberrations of the condenser 20. The light source 18 is focused on the sample 32. Resolving power depends only on the degree of coherence of the light incident upon the object and on the properties of the microscope objective 12. Aberrations of the condenser 20 have no influence on the resolving of a microscope.
FIG. 2b illustrates the Koehler illumination 34 method within a prior art microscope system suitable for Koehler illumination 34. The system includes a light source 18, an auxiliary lens 36, a diaphragm 28, a condenser aperture 26, a condenser 20, a slide 30, a sample 32 coupled to the slide 30, and a microscope objective 12. In this method, the auxiliary lens 36 is placed close to the diaphragm 28 and forms an image of the source light in the focal plane of the condenser 20 which now includes a condenser diaphragm 28. Rays from each light source 18 point then emerge from the condenser 20 as a parallel beam. The light source 18 is focused on an aperture of the condenser 20. Irregularities in the brightness distribution on the source light do not cause irregularities in the intensity of the field illumination.
Furthermore, recent progress in high-resolution optical microscopy has been boosted by demands of cellular biology and nanoscience. In fluorescence microscopy the image resolution of few tens of nanometers has been demonstrated. However, in transmission and reflection microscopy, with visible light illumination even for modern confocal instruments, the reported lateral image resolution does not surpass 180 nm.